The human cytomegalovirus (HCMV) is a large human herpesvirus that latently infects 70-100% of adults in populations worldwide. Though usually asymptomatic in healthy individuals, it can cause severe morbidity and mortality upon primary infection or reactivation in immunocompromised individuals and has been implicated as a cofactor in the progression of certain types of proliferative disease. Several studies have shown that HCMV viral DNA associates with histones and undergoes chromatinization upon entering the nucleus of infected cells and that change in chromatin structure are strongly correlated with changes in viral gene expression. Substantial evidence supports the "Histone Code" theory that specific combinatorial post-translational modifications (PTMs) of the histone proteins on nucleosomes modulate these changes in chromatin structure, which in turn subsequently regulate gene transcription. The goals of this proposal are to identify, quantify, and characterize the dynamic Histone Codes expressed during HCMV lytic infection and their corresponding effects on viral replication. In Specific Aim 1, I will identify and quantify the histone PTMs that are differentially expressed during lytic viral infection in fibroblasts using a Bottom Up LC-MS protocol based on stable-isotope chemical derivatization. Then in Specific Aim 2, I will utilize a novel Middle Down LC-MS/MS approach that was developed for analyzing highly- modified proteins to identify and quantify the global combinatorial Histone Codes expressed during lytic infection in viral and cellular chromatin. The biological roles of the Histone Codes found to be significantly up- or down-regulated in this analysis will be characterized by performing subsequent RNAi knock down experiments and then monitoring the downstream changes in other Histone Codes, gene expression, and virus yield. In Specific Aim 3, I will characterize the genome-specific Histone Codes "read" by nucleosome binding proteins during lytic viral infection using a novel ChIP-Seq MS protocol. In this method, I will FLAG-tag important nucleosome binding proteins (i.e., the "readers" corresponding to Histone Codes that were determined to be significantly regulated in Specific Aim 2) and then enrich for the mononucleosomes bound by these tagged readers using FLAG immunoprecipitation and competitive FLAG peptide elution. The recovered histones will be analyzed by the Middle Down LC-MS/MS approach, characterized by the RNAi knock down method, and then mapped onto to the genome since I can also sequence the DNA associated with the recovered mononucleosomes. Lastly, I will use the standard chromatin immunoprecipitation (ChIP) protocol to investigate the viral and cellular proteins that bind viral DNA directly to modulate viral transcription. To date, no preventative HMCV vaccinations exist and treatment options suffer from low bioavailability, toxicity, and viral resistance. An understanding the epigenetic mechanisms driving viral gene expression would be tremendously beneficial to the design of drug and potential epigenetic therapies. PUBLIC HEALTH RELEVANCE: The majority of adults worldwide are latently infected with the human cytomegalovirus (HMCV), which can cause serious illness and even death in immunocompromised individuals. As preventative HMCV vaccinations do not exist and current treatment options suffer from many drawbacks, I hope to elucidate through this research the underlying epigenetic mechanisms driving viral gene expression in order to assist in the design and development of drug and epigenetic therapies.